Gniadek, Thomas J.

BACKGROUNDSingle antigen bead (SAB) assays are used to identify human leukocyte antigen (HLA) antibodies in patients with platelet refractoriness due to HLA Class I alloimmunization. Some laboratories use serum pretreatment regimens to eliminate interference from immunoglobulin M antibodies and complement. These modifications may contribute to interlaboratory variability, which is a recognized problem with the SAB assay.STUDY DESIGN AND METHODSFive patientsʼ sera were overnight shipped to 12 laboratories in the United States and internationally. Recipients used their labʼs SAB procedure to identify HLA Class I antibodies. The resultant mean fluorescence intensity (MFI) data were compared by instrumentation, bead lot, and pretreatment regimens. Laboratory‐specific cutoffs for positive antibodies were applied to the results.RESULTSInterlaboratory variability for MFI values appears to be associated with different pretreatment regimens. The coefficient of variation (CV) of MFI from samples pretreated with ethylenediaminetetraacetic acid, dithiothreitol, or heat inactivation (EDHI) were similar, ranging from 14% to 56% (mean, 22%). For samples with no pretreatment, the CVs were significantly higher than EDHI‐treated samples, ranging from 25% to 74% (mean, 39%; 95% confidence interval, 12.10‐21.90; p < 0.0001). An intralaboratory comparison of pretreatment regimens confirmed these findings. Some positive antibody specificities present in EDHI‐treated samples were negative in corresponding samples with no pretreatment when laboratory‐specific cutoffs for positive antibodies were applied.CONCLUSIONOur results show that greater interlaboratory precision can be achieved when samples are pretreated with EDHI as opposed to no pretreatment, likely because these pretreatments eliminate interference from inhibitors. Inhibitors may mask antibodies, leading to missed (or uncalled) specificities when no pretreatment is used.

A female presented in the sixth decade of life with a history of undifferentiated small cell carcinoma of the right breast in clinical remission, status-post chemotherapy and resection 6 years previously, presented with a chronic anterior knee skin nodule that grew in size over the prior 5–6 weeks. She had no history of opportunistic infections or recent immunosuppression. As it grew, the nodule became tender to touch. Examination revealed a 4–6 mm superficial purple-red nodule. Also, a similar lesion was present on the dorsum of her left foot for the past year, which also recently grew and became tender. The patient did report frequently kneeling on soil when gardening in Florida. She reported no other symptoms. Due to a concern for cutaneous metastasis of the patient’s previously diagnosed small cell carcinoma of the breast, the anterior knee lesion was biopsied. Histology revealed histocyte-rich inflammation with foci of acute inflammation as well as pigmented fungal forms. Subsequent fungal culture of excised tissue grew Cladophialophora bantiana, identified by ribosomal gene DNA sequencing.